US2023193231A1PendingUtilityA1
Engineered gene-editing proteins
Est. expiryMay 12, 2040(~13.8 yrs left)· nominal 20-yr term from priority
C12N 9/22C12N 5/0629A61F 7/00C12N 15/102
65
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Claims
Abstract
The present invention relates in part to nucleic acids encoding gene editing proteins, including novel engineered variants.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method for gene-editing a cell, comprising
(a) providing a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprising a repeat variable di-residue (RVD) at residue 12 or 13 which targets the DNA-binding domain to a target DNA molecule; and
(ii) a nuclease domain comprising a catalytic domain of a nuclease,
wherein the nucleic acid molecule is a synthetic RNA molecule; and (b) contacting the cell with the one or more synthetic RNA molecules to yield a nick or double-strand break in a target DNA molecule in the cell, wherein the contacting occurs at about 30° C. to about 35° C.
2 . The method of claim 1 , wherein the contacting comprises the cell uptaking the one or more synthetic RNA molecules.
3 . The method of claim 1 or 2 , wherein the contacting comprises transfection.
4 . The method of any one of claims 1 - 3 , wherein the contacting occurs at about 30° C. or about 33° C.
5 . The method of any one of claims 1 - 4 , wherein the gene-editing protein is functionally temperature-switchable.
6 . The method of any one of claims 1 - 5 , wherein the method allows for conditional gene-editing.
7 . The method of any one of claims 1 - 6 , wherein the method is conducted in vitro.
8 . The method of any one of claims 1 - 6 , wherein the method is conducted ex vivo.
9 . The method of any one of claims 1 - 8 , further comprising the step of (c) culturing the contacted cell at about 30° C. to about 35° C., optionally about 33° C.
10 . The method of any one of claims 1 - 8 , further comprising the step of (c) culturing the contacted cell at about 30° C. or about 33° C.
11 . The method of any one of claims 1 - 10 , further comprising the step of (c) administering the contacted cell to a subject in need thereof.
12 . The method of any one of claims 1 - 11 , wherein the cell is formulated for therapeutic use.
13 . The method of any one of claims 1 - 12 , wherein the cell is suitable for administration to a human subject.
14 . The method of any one of claims 1 - 6 and 9 - 13 , wherein the method is conducted in vivo.
15 . The method of any one of claims 1 - 14 , wherein the method comprises reducing the body temperature of a subject, optionally via whole-body hypothermia.
16 . The method of any one of claims 1 - 14 , wherein the method comprises applying one or more cooling elements to a cell or tissue in vivo to reduce temperature, the cooling element optionally being a cryocompression device.
17 . The method of any one of claims 1 - 16 , wherein the cell is of the integumentary system.
18 . The method of any one of claims 1 - 17 , wherein the cell is a skin cell.
19 . The method of claim 18 , wherein the skin cell is a fibroblast, a keratinocyte, a melanocyte, or an adipocyte.
20 . A method of treating a disease or disorder comprising
(a) contacting a cell or tissue with one or more cooling elements to locally reduce temperature; and (b) administering a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13 which targets the DNA-binding domain to a target DNA molecule; and
(ii) a nuclease domain comprising a catalytic domain of a nuclease,
wherein the nucleic acid molecule is a synthetic RNA molecule.
21 . The method of claim 20 , wherein the cell or tissue is of the integumentary system.
22 . The method of claim 20 or 21 , wherein the cell or tissue is a skin cell.
23 . The method of claim 22 , wherein the skin cell is a fibroblast, a keratinocyte, a melanocyte, or an adipocyte.
24 . A method of treating a disease or disorder comprising
(a) providing a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13 which targets the DNA-binding domain to a target DNA molecule; and
(ii) a nuclease domain comprising a catalytic domain of a nuclease,
wherein the nucleic acid molecule is a synthetic RNA molecule; (b) transfecting the cell with the one or more synthetic RNA molecules to yield a nick or double-strand break in a target DNA molecule in the cell, wherein the transfecting occurs at about 30° C. to about 35° C.; and (c) administering the transfected cell to a subject in need thereof.
25 . The method of any one of claims 1 - 24 , wherein the nuclease domain is capable of forming a dimer with another nuclease domain.
26 . The method of any one of claims 1 - 25 , wherein the catalytic domain is from FokI, StsI, or a hybrid thereof.
27 . The method of any one of claims 1 - 26 , wherein the gene-editing protein is capable of generating a nick or double-strand break in a target DNA molecule.
28 . The method of any one of claims 1 - 27 , wherein the RVD recognizes one base pair in the nucleic acid molecule.
29 . The method of any one of claims 1 - 28 , wherein the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(null), HA, ND, and HI.
30 . The method of any one of claims 1 - 29 , wherein the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA.
31 . The method of any one of claims 1 - 30 , wherein the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS.
32 . The method of any one of claims 1 - 31 , wherein the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(null), and IG
33 . The method of any one of claims 1 - 32 , wherein the repeat sequence is 33 or 34 amino acids long.
34 . The method of any one of claims 1 - 32 , wherein the repeat sequence is 36-39 amino acids long.
35 . The method of any one of claims 1 - 34 , wherein the DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyz (SEQ ID NO: 12), wherein:
v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, and z is GGRPALE (SEQ ID NO: 1), GGKQALE (SEQ ID NO: 2), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 3), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 5), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 6), GGKQALETVQRLLPVLCQD (SEQ ID NO: 7) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 8).
36 . The method of any one of claims 1 - 34 , wherein at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 11), wherein:
v is Q, D or E,
w is S or N,
x is I, H, N, or I,
y is D, A, I, N, H, K, S, G or null,
z is GGRPALE (SEQ ID NO: 1), GGKQALE (SEQ ID NO: 2), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 3), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 5), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 6), GGKQALETVQRLLPVLCQD (SEQ ID NO: 7) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 8), and
α is any four consecutive amino acids.
37 . The method of claim 36 , wherein α comprises at least one glycine (G) residue.
38 . The method of claim 36 or 37 , wherein α comprises at least one histidine (H) residue.
39 . The method of any one of claims 36 - 38 , wherein α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35.
40 . The method of any one of claims 36 - 39 , wherein α comprises at least one aspartic acid (D) residue.
41 . The method of any one of claims 36 - 40 , wherein α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.
42 . The method of any one of claims 36 - 41 , wherein α comprises one or more hydrophilic residues, optionally selected from:
a polar and positively charged hydrophilic amino acid, optionally selected from arginine (R) and lysine (K);
a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C);
a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glutamate (E), and
an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).
43 . The method of any one of claims 36 - 42 , wherein α comprises one or more hydrophobic residues, optionally selected from:
a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and
a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
44 . The method of any one of claims 36 - 43 , wherein α is selected from GHGG (SEQ ID NO: 38), HGSG (SEQ ID NO: 39), HGGG (SEQ ID NO: 40), GGHD (SEQ ID NO: 41), GAHD (SEQ ID NO: 42), AHDG (SEQ ID NO: 43), PHDG (SEQ ID NO: 44), GPHD (SEQ ID NO: 45), GHGP (SEQ ID NO: 46), PHGG (SEQ ID NO: 47), PHGP (SEQ ID NO: 48, AHGA (SEQ ID NO: 49), LHGA (SEQ ID NO: 50), VHGA (SEQ ID NO: 51), IVHG (SEQ ID NO: 52), IHGM (SEQ ID NO: 53), RHGD (SEQ ID NO: 54), RDHG (SEQ ID NO: 55), RHGE (SEQ ID NO: 56), HRGE (SEQ ID NO: 57), HRGD (SEQ ID NO: 58), GPYE (SEQ ID NO: 59), NHGG (SEQ ID NO: 60), THGG (SEQ ID NO: 61), GTHG (SEQ ID NO: 62), GSGS (SEQ ID NO: 63), GSGG (SEQ ID NO: 64), GGGG (SEQ ID NO: 65), GRGG (SEQ ID NO: 66), and GKGG (SEQ ID NO: 67).
45 . The method of any one of claims 1 - 44 , wherein the synthetic RNA molecule is mRNA.
46 . The method of any one of claims 1 - 45 , wherein the synthetic RNA molecule is in vitro transcribed.
47 . The method of any one of claims 1 - 46 , wherein the synthetic RNA molecule comprises one or more non-canonical nucleotides.
48 . The method of any one of claims 45 - 47 , wherein the mRNA comprises one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.
49 . The method of any one of claims 47 - 48 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the non-canonical nucleotides comprises one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.
50 . The method of any one of claims 1 - 49 , wherein the synthetic RNA molecule comprises a 5′ cap structure.
51 . The method of any one of claims 1 - 50 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a Kozak consensus sequence.
52 . The method of any one of claims 1 - 51 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a sequence that increases RNA stability in vivo.
53 . The method of any one of claims 1 - 52 , wherein the synthetic RNA molecule comprises a 3′-UTR comprising a sequence that increases RNA stability in vivo.
54 . The method of any one of claims 51 - 52 , wherein the 5′-UTR comprises an alpha-globin or beta-globin 5′-UTR sequence and/or wherein the 3′-UTR comprises an alpha-globin or beta-globin 3′-UTR sequence.
55 . The method of any one of claims 1 - 54 , wherein the synthetic RNA molecule comprises a 3′ poly(A) tail or a tail comprising of a plurality of adenines with one or more guanines.
56 . The method of any one of claims 1 - 55 , wherein the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
57 . The method of any one of claims 1 - 55 , wherein the method further comprises administering a linear DNA repair template or contacting the cell a linear DNA repair template.
58 . A method for making a targeted composition for gene-editing, comprising:
(a) selecting a DNA target sequence, the DNA target sequence being substantially unmethylated; and (b) constructing a nucleic acid encoding a gene-editing protein, the gene-editing protein being designed to specifically target the substantially unmethylated DNA target sequence and comprising:
(i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 11) and is between 36 and 39 amino acids long, wherein:
v is Q, D or E,
w is S or N,
x is I, H, N, or I,
y is D, A, I, N, H, K, S, G or null,
z is GGRPALE (SEQ ID NO: 1), GGKQALE (SEQ ID NO: 2), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 3), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 5), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 6), GGKQALETVQRLLPVLCQD (SEQ ID NO: 7) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 8),
α is four consecutive amino acids;
(ii) a nuclease domain comprising a catalytic domain of a nuclease,
wherein the nucleic acid molecule is a synthetic RNA molecule.
59 . The method of claim 58 , wherein the identity of x and y are based on the substantially unmethylated DNA target sequence.
60 . The method of claim 58 or 59 , wherein x and y:
recognize a C residue in the nucleic acid molecule and are selected from HD, N(null), HA, ND, and HI;
recognize a G residue in the nucleic acid molecule and are selected from NN, NH, NK, HN, and NA;
recognize an A residue in the nucleic acid molecule and are selected from NI and NS; or
recognize a T residue in the nucleic acid molecule and are selected from NG, HG, H(null), and IG.
61 . The method of any one of claims 58 - 60 , wherein α comprises at least one glycine (G) residue.
62 . The method of any one of claims 58 - 61 , wherein α comprises at least one histidine (H) residue.
63 . The method of any one of claims 58 - 62 , wherein α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35.
64 . The method of any one of claims 58 - 63 , wherein α comprises at least one aspartic acid (D) residue.
65 . The method of any one of claims 58 - 64 , wherein α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.
66 . The method of any one of claims 58 - 65 , wherein α comprises one or more hydrophilic residues, optionally selected from:
a polar and positively charged hydrophilic amino acid, optionally selected from arginine (R) and lysine (K);
a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C);
a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glutamate (E), and
an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).
67 . The method of any one of claims 58 - 66 , wherein α comprises one or more hydrophobic residues, optionally selected from:
a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and
a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
68 . The method of any one of claims 58 - 67 , wherein α is selected from GHGG (SEQ ID NO: 38), HGSG (SEQ ID NO: 39), HGGG (SEQ ID NO: 40), GGHD (SEQ ID NO: 41), GAHD (SEQ ID NO: 42), AHDG (SEQ ID NO: 43), PHDG (SEQ ID NO: 44), GPHD (SEQ ID NO: 45), GHGP (SEQ ID NO: 46), PHGG (SEQ ID NO: 47), PHGP (SEQ ID NO: 48, AHGA (SEQ ID NO: 49), LHGA (SEQ ID NO: 50), VHGA (SEQ ID NO: 51), IVHG (SEQ ID NO: 52), IHGM (SEQ ID NO: 53), RHGD (SEQ ID NO: 54), RDHG (SEQ ID NO: 55), RHGE (SEQ ID NO: 56), HRGE (SEQ ID NO: 57), HRGD (SEQ ID NO: 58), GPYE (SEQ ID NO: 59), NHGG (SEQ ID NO: 60), THGG (SEQ ID NO: 61), GTHG (SEQ ID NO: 62), GSGS (SEQ ID NO: 63), GSGG (SEQ ID NO: 64), GGGG (SEQ ID NO: 65), GRGG (SEQ ID NO: 66), and GKGG (SEQ ID NO: 67).
69 . The method of any one of claims 58 - 68 , wherein the synthetic RNA molecule is mRNA.
70 . The method of any one of claims 58 - 69 , wherein the synthetic RNA molecule is in vitro transcribed.
71 . The method of any one of claims 58 - 70 , wherein the synthetic RNA molecule comprises one or more non-canonical nucleotides.
72 . The method of any one of claims 69 - 71 , wherein the mRNA comprises one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.
73 . The method of any one of claims 70 - 71 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the non-canonical nucleotides comprises one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.
74 . The method of any one of claims 58 - 73 , wherein the synthetic RNA molecule comprises a 5′ cap structure.
75 . The method of any one of claims 58 - 74 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a Kozak consensus sequence.
76 . The method of any one of claims 58 - 75 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a sequence that increases RNA stability in vivo.
77 . The method of any one of claims 58 - 76 , wherein the synthetic RNA molecule comprises a 3′-UTR comprising a sequence that increases RNA stability in vivo.
78 . The method of any one of claims 75 - 76 , wherein the 5′-UTR comprises an alpha-globin or beta-globin 5′-UTR sequence.
79 . The method of claim 77 , wherein the 3′-UTR comprises an alpha-globin or beta-globin 3′-UTR sequence.
80 . The method of any one of claims 58 - 79 , wherein the synthetic RNA molecule comprises a 3′ poly(A) tail or a tail comprising of a plurality of adenines with one or more guanines.
81 . The method of any one of claims 58 - 80 , wherein the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
82 . A method for gene-editing a cell, comprising
(a) providing a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 11) and is between 36 and 39 amino acids long, wherein:
v is Q, D or E,
w is S or N,
x is I, H, N, or I,
y is D, A, I, N, H, K, S, G or null,
z is GGRPALE (SEQ ID NO: 1), GGKQALE (SEQ ID NO: 2), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 3), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 5), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 6), GGKQALETVQRLLPVLCQD (SEQ ID NO: 7) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 8),
α is four consecutive amino acids;
(ii) a nuclease domain comprising a catalytic domain of a nuclease,
wherein the nucleic acid molecule is a synthetic RNA molecule; and (b) contacting the cell with the one or more synthetic RNA molecules yield a nick or double-strand break in a target DNA molecule in the cell; and (c) contacting the cell with a demethylating agent.
83 . The method of claim 82 , wherein the demethylating agent is selected from 5-azacitidine and 5-aza-2′-deoxycitidine (decitabine).
84 . The method of claim 82 or 83 , wherein the method allows for conditional gene-editing.
85 . The method of any one of claims 82 - 84 , wherein the method is conducted in vitro.
86 . The method of any one of claims 82 - 84 , wherein the method is conducted ex vivo.
87 . The method of any one of claims 82 - 86 , further comprising the step of (c) administering the contacted cell to a subject in need thereof.
88 . The method of any one of claims 82 - 87 , wherein the cell is formulated for therapeutic use.
89 . The method of any one of claims 82 - 88 , wherein the cell is suitable for administration to a human subject.
90 . The method of any one of claims 82 - 84 and 87 - 89 , wherein the method is conducted in vivo.
91 . The method of any one of claims 82 - 90 , wherein α comprises at least one glycine (G) residue.
92 . The method of any one of claims 82 - 91 , wherein α comprises at least one histidine (H) residue.
93 . The method of any one of claims 82 - 92 , wherein α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35.
94 . The method of any one of claims 82 - 93 , wherein α comprises at least one aspartic acid (D) residue.
95 . The method of any one of claims 82 - 94 , wherein α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.
96 . The method of any one of claims 82 - 95 , wherein α comprises one or more hydrophilic residues, optionally selected from:
a polar and positively charged hydrophilic amino acid, optionally selected from arginine (R) and lysine (K);
a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C);
a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glutamate (E), and
an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).
97 . The method of any one of claims 82 - 96 , wherein α comprises one or more hydrophobic residues, optionally selected from:
a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and
a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
98 . The method of any one of claims 82 - 97 , wherein α is selected from GHGG (SEQ ID NO: 38), HGSG (SEQ ID NO: 39), HGGG (SEQ ID NO: 40), GGHD (SEQ ID NO: 41), GAHD (SEQ ID NO: 42), AHDG (SEQ ID NO: 43), PHDG (SEQ ID NO: 44), GPHD (SEQ ID NO: 45), GHGP (SEQ ID NO: 46), PHGG (SEQ ID NO: 47), PHGP (SEQ ID NO: 48, AHGA (SEQ ID NO: 49), LHGA (SEQ ID NO: 50), VHGA (SEQ ID NO: 51), IVHG (SEQ ID NO: 52), IHGM (SEQ ID NO: 53), RHGD (SEQ ID NO: 54), RDHG (SEQ ID NO: 55), RHGE (SEQ ID NO: 56), HRGE (SEQ ID NO: 57), HRGD (SEQ ID NO: 58), GPYE (SEQ ID NO: 59), NHGG (SEQ ID NO: 60), THGG (SEQ ID NO: 61), GTHG (SEQ ID NO: 62), GSGS (SEQ ID NO: 63), GSGG (SEQ ID NO: 64), GGGG (SEQ ID NO: 65), GRGG (SEQ ID NO: 66), and GKGG (SEQ ID NO: 67).
99 . The method of any one of claims 82 - 98 , wherein the synthetic RNA molecule is mRNA.
100 . The method of any one of claims 82 - 99 , wherein the synthetic RNA molecule is in vitro transcribed.
101 . The method of any one of claims 82 - 100 , wherein the synthetic RNA molecule comprises one or more non-canonical nucleotides.
102 . The method of any one of claims 98 - 100 , wherein the mRNA comprises one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza azaguanosine.
103 . The method of any one of claims 101 - 102 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the non-canonical nucleotides comprises one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.
104 . The method of any one of claims 82 - 103 , wherein the synthetic RNA molecule comprises a 5′ cap structure.
105 . The method of any one of claims 82 - 104 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a Kozak consensus sequence.
106 . The method of any one of claims 82 - 105 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a sequence that increases RNA stability in vivo.
107 . The method of any one of claims 82 - 106 , wherein the synthetic RNA molecule comprises a 3′-UTR comprising a sequence that increases RNA stability in vivo.
108 . The method of any one of claims 105 - 106 , wherein the 5′-UTR comprises an alpha-globin or beta-globin 5′-UTR sequence or the 3′-UTR comprises an alpha-globin or beta-globin 3′-UTR sequence.
109 . The method of claim 107 , wherein the synthetic RNA molecule comprises a 3′ poly(A) tail or a tail comprising of a plurality of adenines with one or more guanines.
110 . The method of any one of claims 82 - 109 , wherein the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
111 . The method of any one of claims 82 - 110 , wherein the method further comprises administering a linear DNA repair template or contacting the cell a linear DNA repair template.
112 . A cell comprising the composition of any one of claims 82 - 111 .
113 . A composition comprising a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(a) the DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13; and (b) the nuclease domain comprising a catalytic domain, the catalytic domain comprising a hybrid of the catalytic domains of FokI and StsI, comprising the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of FokI with at least one of the domains of FokI being substituted in whole or in part with the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of StsI and optionally comprising at least one mutation.
114 . The composition of claim 113 , wherein the catalytic domain comprises:
α1, α2, α3, and α6 of FokI, α1, α2, α3, of FokI and α6 of StsI, α1 and α2 of FokI and α3 and α6 of StsI, α1 of FokI and α2, α3 and α6 of StsI, α1 of StsI and α2, α3 and α6 of FokI, α1 and α2 of StsI and α3 and α6 of FokI, α1, α2, α3, of StsI and α6 of FokI, or α1, α2, α3, and α6 of StsI.
115 . The composition of claim 113 , wherein the catalytic domain comprises:
α4 and α6 of FokI, α4 of FokI and α6 of StsI, α4 of StsI and α6 of FokI, or α4 and α6 of StsI.
116 . The composition of any one of claims 113 - 115 , wherein the catalytic domains of FokI and StsI comprises the amino acids sequences of FIG. 6 or Table 1, or a sequence having at least about 95%, or at least about 97%, or at least about 98% identity thereto.
117 . The composition of any one of claims 113 - 116 , wherein the catalytic domain comprises one or more amino acid mutations, optionally selected from substitutions, insertions, deletions, and truncations, or combinations thereof.
118 . The composition of any one of claims 113 - 117 , wherein the nuclease domain is capable of forming a dimer with another nuclease domain.
119 . The composition of any one of claims 113 - 118 , wherein the gene-editing protein is capable of generating a nick or double-strand break in a target DNA molecule.
120 . The composition of any one of claims 113 - 119 , wherein the RVD recognizes one base pair in the nucleic acid molecule.
121 . The composition of any one of claims 113 - 120 , wherein the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(null), HA, ND, and HI.
122 . The composition of any one of claims 113 - 121 , wherein the RVD recognizes a G residue in the nucleic acid molecule and is selected from NN, NH, NK, HN, and NA.
123 . The composition of any one of claims 113 - 122 , wherein the RVD recognizes an A residue in the nucleic acid molecule and is selected from NI and NS.
124 . The composition of any one of claims 113 - 123 , wherein the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(null), and IG.
125 . The composition of any one of claims 113 - 124 , wherein the repeat sequence is 33 or 34 amino acids long.
126 . The composition of any one of claims 113 - 124 , wherein the repeat sequence is 36-39 amino acids long.
127 . The composition of any one of claims 113 - 126 , wherein the DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyz (SEQ ID NO: 12), wherein:
v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, and z is GGRPALE (SEQ ID NO: 1), GGKQALE (SEQ ID NO: 2), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 3), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 5), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 6), GGKQALETVQRLLPVLCQD (SEQ ID NO: 7) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 8).
128 . The composition of any one of claims 113 - 126 , wherein at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 11), wherein:
v is Q, D or E,
w is S or N,
x is I, H, N, or I,
y is D, A, I, N, H, K, S, G or null,
z is GGRPALE (SEQ ID NO: 1), GGKQALE (SEQ ID NO: 2), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 3), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 5), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 6), GGKQALETVQRLLPVLCQD (SEQ ID NO: 7) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 8), and
α is any four consecutive amino acids.
129 . The composition of claim 128 , wherein α comprises at least one glycine (G) residue.
130 . The composition of any one of claims 128 - 129 , wherein α comprises at least one histidine (H) residue.
131 . The composition of any one of claims 128 - 130 , wherein α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35.
132 . The composition of any one of claims 128 - 131 , wherein α comprises at least one aspartic acid (D) residue.
133 . The composition of any one of claims 128 - 132 , wherein α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.
134 . The composition of any one of claims 128 - 133 , wherein α comprises one or more hydrophilic residues, optionally selected from:
a polar and positively charged hydrophilic amino acid, optionally selected from arginine (R) and lysine (K);
a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C);
a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glutamate (E), and
an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).
135 . The composition of any one of claims 128 - 134 , wherein α comprises one or more hydrophobic residues, optionally selected from:
a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and
a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
136 . The composition of any one of claims 128 - 135 , wherein α is selected from GHGG (SEQ ID NO: 38), HGSG (SEQ ID NO: 39), HGGG (SEQ ID NO: 40), GGHD (SEQ ID NO: 41), GAHD (SEQ ID NO: 42), AHDG (SEQ ID NO: 43, PHDG (SEQ ID NO: 44), GPHD (SEQ ID NO: 45), GHGP (SEQ ID NO: 46), PHGG (SEQ ID NO: 47), PHGP (SEQ ID NO: 48), AHGA (SEQ ID NO: 49), LHGA (SEQ ID NO: 50), VHGA (SEQ ID NO: 51), IVHG (SEQ ID NO: 52), IHGM (SEQ ID NO: 53), RHGD (SEQ ID NO: 54), RDHG (SEQ ID NO: 55), RHGE (SEQ ID NO: 56), HRGE (SEQ ID NO: 57, HRGD (SEQ ID NO: 58), GPYE (SEQ ID NO: 59), NHGG (SEQ ID NO: 60), THGG (SEQ ID NO: 61), GTHG (SEQ ID NO: 62), GSGS (SEQ ID NO: 63), GSGG (SEQ ID NO: 64), GGGG (SEQ ID NO: 65), GRGG (SEQ ID NO: 66), and GKGG (SEQ ID NO: 67).
137 . The composition of any one of claims 113 - 136 , wherein the nucleic acid is a synthetic RNA molecule.
138 . The method of any one of claims 113 - 137 , wherein the synthetic RNA molecule is mRNA.
139 . The composition of any one of claims 113 - 138 , wherein the synthetic RNA molecule is in vitro transcribed.
140 . The composition of any one of claims 113 - 139 , wherein the synthetic RNA molecule comprises one or more non-canonical nucleotides.
141 . The composition of any one of claims 138 - 140 , wherein the mRNA comprises one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.
142 . The composition of any one of claims 140 - 141 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the non-canonical nucleotides comprises one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.
143 . The composition of any one of claims 113 - 142 , wherein the synthetic RNA molecule comprises a 5′ cap structure.
144 . The composition of any one of claims 113 - 143 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a Kozak consensus sequence.
145 . The composition of any one of claims 113 - 144 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a sequence that increases RNA stability in vivo.
146 . The composition of any one of claims 113 - 145 , wherein the synthetic RNA molecule comprises a 3′-UTR comprising a sequence that increases RNA stability in vivo.
147 . The composition of any one of claims 144 - 145 , wherein the 5′-UTR comprises an alpha-globin or beta-globin 5′-UTR sequence or wherein the 3′-UTR comprises an alpha-globin or beta-globin 3′-UTR sequence.
148 . The composition of any one of claims 113 - 147 , wherein the synthetic RNA molecule comprises a 3′ poly(A) tail or a tail comprising of a plurality of adenines with one or more guanines.
149 . The composition of any one of claims 113 - 148 , wherein the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
150 . The composition of any one of claims 113 - 149 , further comprising a linear DNA repair template or contacting the cell a linear DNA repair template.
151 . A cell comprising the composition of any one of claims 113 - 150 .
152 . A composition comprising a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(a) the DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα (SEQ ID NO: 11) and is between 36 and 39 amino acids long, wherein:
v is Q, D or E,
w is S or N,
x is I, H, N, or I,
y is D, A, I, N, H, K, S, G or null,
z is GGRPALE (SEQ ID NO: 1), GGKQALE (SEQ ID NO: 2), GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 3), GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 4), GKQALETVQRLLPVLCQDHG (SEQ ID NO: 5), GKQALETVQRLLPVLCQAHG (SEQ ID NO: 6), GGKQALETVQRLLPVLCQD (SEQ ID NO: 7) or GGKQALETVQRLLPVLCQA (SEQ ID NO: 8),
α is four consecutive amino acids, with the proviso that α is not GHGG (SEQ ID NO: 38), HGSG (SEQ ID NO: 39), and HGGG (SEQ ID NO: 40); and
(b) the nuclease domain comprising a catalytic domain of a nuclease.
153 . A composition comprising a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(a) the DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises an amino acid sequence of between 36 and 39 amino acids long, of which the four most C-terminal amino acids are selected from GGHD (SEQ ID NO: 41), GAHD (SEQ ID NO: 42), AHDG (SEQ ID NO: 43), PHDG (SEQ ID NO: 44), GPHD (SEQ ID NO: 45), GHGP (SEQ ID NO: 46), PHGG (SEQ ID NO: 47), PHGP (SEQ ID NO: 48), AHGA (SEQ ID NO: 49), LHGA (SEQ ID NO: 50), VHGA (SEQ ID NO: 51, IVHG (SEQ ID NO: 52), IHGM (SEQ ID NO: 53), RHGD (SEQ ID NO: 54), RDHG (SEQ ID NO: 55), RHGE (SEQ ID NO: 56), HRGE (SEQ ID NO: 57), HRGD (SEQ ID NO: 58), GPYE (SEQ ID NO: 59), NHGG (SEQ ID NO: 60), THGG (SEQ ID NO: 61), GTHG (SEQ ID NO: 62), GSGS (SEQ ID NO: 63), GSGG (SEQ ID NO: 64), GGGG (SEQ ID NO: 65), GRGG (SEQ ID NO: 66), and GKGG (SEQ ID NO: 67); and (b) the nuclease domain comprises a catalytic domain of a nuclease.
154 . The composition of claim 152 , wherein α comprises at least one glycine (G) residue.
155 . The composition of claim 152 or claim 154 , wherein α comprises at least one histidine (H) residue.
156 . The composition of any one of claims 152 and 154 - 155 , wherein α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35.
157 . The composition of any one of claims 152 and 154 - 156 , wherein α comprises at least one aspartic acid (D) residue.
158 . The composition of any one of claims 152 and 154 - 157 , wherein α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.
159 . The composition of any one of claims 152 and 154 - 158 , wherein α comprises one or more hydrophilic residues, optionally selected from:
a polar and positively charged hydrophilic amino acid, optionally selected from arginine (R) and lysine (K);
a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cysteine (C);
a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glutamate (E), and
an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).
160 . The composition of any one of claims 152 and 154 - 159 , wherein α comprises one or more hydrophobic residues, optionally selected from:
a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and
a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y).
161 . The composition of any one of claims 152 - 160 , wherein the nuclease domain is capable of forming a dimer with another nuclease domain.
162 . The composition of any one of claims 152 - 161 , wherein the nuclease is selected from FokI, StsI, or a hybrid thereof.
163 . The composition of any one of claims 152 - 162 , wherein the gene-editing protein is capable of generating a nick or double-strand break in a target DNA molecule.
164 . The composition of any one of claims 152 - 163 , wherein the nucleic acid is a synthetic RNA molecule.
165 . The method of claim 164 , wherein the synthetic RNA molecule is mRNA.
166 . The composition of claim 164 or 165 , wherein the synthetic RNA molecule is in vitro transcribed.
167 . The composition of any one of claims 164 - 166 , wherein the synthetic RNA molecule comprises one or more non-canonical nucleotides.
168 . The composition of any one of claims 165 - 167 , wherein the mRNA comprises one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl methylpseudoisocytidine, N4-methyl-5-aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino azacytidine, N4-aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5-hydroxy azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy amino-5-azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7-deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8-azaguanosine.
169 . The composition of any one of claims 167 - 168 , wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the non-canonical nucleotides comprises one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.
170 . The composition of any one of claims 164 - 169 , wherein the synthetic RNA molecule comprises a 5′ cap structure.
171 . The composition of any one of claims 164 - 170 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a Kozak consensus sequence.
172 . The composition of any one of claims 164 - 171 , wherein the synthetic RNA molecule comprises a 5′-UTR comprising a sequence that increases RNA stability in vivo.
173 . The composition of any one of claims 164 - 172 , wherein the synthetic RNA molecule comprises a 3′-UTR comprising a sequence that increases RNA stability in vivo.
174 . The composition of any one of claims 171 - 172 , wherein the 5′-UTR comprises an alpha-globin or beta-globin 5′-UTR sequence.
175 . The composition of claim 173 , wherein the 3′-UTR comprises an alpha-globin or beta-globin 3′-UTR sequence
176 . The composition of any one of claims 164 - 175 , wherein the synthetic RNA molecule comprises a 3′ poly(A) tail or a tail comprising of a plurality of adenines with one or more guanines.
177 . The composition of any one of claims 152 - 176 , wherein at least one of the repeat sequences contains a region capable of binding to a binding site in a target DNA molecule, the binding site containing a defined sequence of between 1 and 5 bases in length.
178 . The composition of any one of claims 152 - 177 , wherein the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.
179 . The composition of any one of claims 152 - 178 , further comprising a linear DNA repair template or contacting the cell a linear DNA repair template.
180 . A composition comprising a nucleic acid encoding a gene-editing protein, the gene-editing protein comprising:
(a) the DNA-binding domain comprises a plurality of repeat sequences and at least one of the repeat sequences comprises an amino acid sequence with one or more substitutions and/or additions in the in the N-terminal region to remove a T 0 dependency; and (b) the nuclease domain comprises a catalytic domain of a nuclease.
181 . The composition of claim 180 , wherein the DNA-binding domain comprises a sequence of Asp225-IVGVGKQWSGARAL-Glu240 (SEQ ID NO: 29), wherein KQWS is replaced with one or more amino acids, e.g. about 2-10 amino acids, or about 4-10 amino acids, or about 6-10 amino acids, or about 8-10 amino acids, or about 4 amino acids, or about 6 amino acids, or about 8 amino acids, or about 10 amino acids.
182 . The composition of claim 180 or 181 , wherein the DNA-binding domain comprises a sequence Asp225-IVGVGGSKRGAGSGARAL-Glu244 (SEQ ID NO: 31).
183 . A method for gene-editing a cell, comprising contacting the cell with the composition of any one of claims 180 - 182 .Join the waitlist — get patent alerts
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